Topoisomerase I suppresses genomic instability by preventing interference between replication and transcription

Topoisomerase I (Top1) is a key enzyme acting at the interface between DNA replication, transcription and mRNA maturation. Here, we show that Top1 suppresses genomic instability in mammalian cells by preventing conflicts between transcription and DNA replication. Using DNA combing and ChIP-on-chip, we found that Top1-deficient cells accumulate stalled replication forks and chromosome breaks in S phase and that breaks occur preferentially at gene-rich regions of the genome. Strikingly, these phenotypes were suppressed by preventing the formation of RNA-DNA hybrids (R-loops) during transcription. Moreover, these defects could be mimicked by depletion of the splicing factor ASF/SF2, which interacts functionally with Top1. Taken together, these data indicate that Top1 prevents replication fork collapse by suppressing the formation of R-loops in an ASF/SF2-dependent manner. We propose that interference between replication and transcription represents a major source of spontaneous replication stress, which could drive genomic instability during early stages of tumorigenesis. Bed files contain the gamma-H2AX enrichment sites described in the paper Gamma-H2AX ChIPed vs. Input DNA in HCT116 shTop1 and shCtrl cells

拓扑异构酶I（Topoisomerase I，简称Top1）是一类关键酶，参与DNA复制、转录与mRNA成熟过程的交叉调控。本研究证实，Top1可通过阻断转录与DNA复制之间的冲突，抑制哺乳动物细胞中的基因组不稳定现象。本研究采用DNA拉伸纤维技术（DNA combing）与染色质免疫共沉淀-芯片（ChIP-on-chip）技术，发现Top1缺陷细胞在S期会积累停滞的复制叉与染色体断裂，且断裂位点优先富集于基因组的基因密集区域。值得注意的是，通过阻断转录过程中RNA-DNA杂交体（R-loops）的形成，可有效抑制上述表型。此外，敲低与Top1存在功能互作的剪接因子ASF/SF2，可模拟上述缺陷表型。综合上述实验结果，本研究表明Top1可通过ASF/SF2依赖的机制抑制R-loops的形成，从而阻止复制叉崩解。本研究提出，复制与转录之间的干扰是自发性复制应激的主要来源，该过程可在肿瘤发生早期驱动基因组不稳定的产生。本研究涉及的BED格式文件（Bed files）包含论文中所述的γ-H2AX富集位点，对应HCT116细胞中shTop1与shCtrl组的γ-H2AX染色质免疫共沉淀样本与Input DNA的富集分析结果。